Immuno-potentiating systems for preparation of immunogenic materials

ABSTRACT

The invention is directed to improved immunopotentiating systems for preparation of immunogenic materials. More particularly, the invention is directed to immunogenic compositions containing a protein, polypeptide, or peptide, a hydrophobic anchor, and a proteosome. The immunogenic compositions are suitable for use as therapeutic agents and vaccines.

FIELD OF THE INVENTION

[0001] This invention relates to means for improving immunogenicproperties of peptides, polypeptides, and proteins by coupling with ahydrophobic anchor which may, in turn, be attached to proteosomes.

SUMMARY OF THE INVENTION

[0002] The instant invention provides an immunopotentiating system forenhancing immunogenic properties of peptides, polypeptides and protein.In some instances, amino acid sequences which have not been consideredimmunogenic or only weakly immunogenic may be rendered effectivelyimmunogenic thereby. The synthesis of amino acid sequences is frequentlyeconomically advantageous over use of the natural antigen. Furthermore,proteins and peptides produced by genetic engineering which do notpossess sufficient antigenicity may effect greater immune reaction whencomplexed with a hydrophobic anchor or “foot” attached to the amino acidsequences through one or more cysteines followed by dimerization orcyclization to form an enhanced peptide or protein structure which canbe complexed with proteosomes. The resulting construct provides animmunogenic peptide comprising (a) peptides, protein fragment, andproteins having bonded thereto (b) a hydrophobic foot attached throughcysteine(s) to the sequence to be render immunogenic, and wherein thehydrophobic foot is complexed to (c) a proteosome. “Proteins”, in thoseinstances when further definition is provided, should be interpreted toinclude polypeptides and protein fragments as well as entire proteins.

[0003] In a preferred embodiment, a method for production of immunogenicpeptides comprises the steps of:

[0004] (a) replicating a core peptide or protein;

[0005] (b) reacting cysteine with the replicated peptide/protein to addat least one cysteine residue to said replicated peptide;

[0006] (c) reacting the replicated peptide/protein-cysteine with analiphatic carboxylic acid or a hydrophobic peptide to add a hydrophobicfoot to said replicated peptide/protein-cysteine to form an enhancedpeptide structure;

[0007] (d) forming disulfide bonds in the enhanced peptide/proteinstructure to effect dimerization or cyclization of said enhanced peptidestructure; and

[0008] (e) complexing the enhanced peptide/protein structure with aproteosome.

[0009] The constructs of the invention may be used for vaccines toprotect against or treat disease conditions such as infectious diseases,malignancies, and toxic effects of chemicals and biologicals. Vaccinesmay also be used to prevent pregnancy.

[0010] Proteosomes are hydrophobic membranous, multimolecular membraneproteins. They may be obtained from any of a number of differentorganisms. Coupling may be accomplished by dialysis or lyophilization.

BACKGROUND OF THE INVENTION

[0011] The development of peptide subunits or recombinant proteinvaccines to protect against pathogenic microorganisms and, of late,malignancies has been impeded by lack of sufficient immunogenicity inthe peptides and proteins produced. Often the untoward effects resultingfrom exposure to an immunogen must be weighed against the adequacy ofimmunogenic properties. The presentation of small peptides, polypeptidesand protein fragments to enhance immunogenicity without increasingundesired side effects from exposure to peptides, and protein fragmentsis an important area of investigation. There exists a paucity ofcarriers and adjuvants that are non-toxic and non-pyrogenic for humanuse. Furthermore, carriers that are safe for human use frequently cannotbe efficiently complexed to the peptides to render them immunogenicwithout altering the proteins.

[0012] The development of substitutes for the whole organism or largeproteins therefrom as vaccines is an important advance in biotechnology.Advances in biotechnology have made it increasingly possible to producevaccines composed of amino acid sequences identical to conserved proteinregions common to many strains of pathogens that may elicitcross-reacting antibodies. If the antigenicity can be improved bycoupling with fragments that increase immunogenicity, improved vaccinescan be developed.

[0013] Prior art of interest includes work of several researchers.Zollinger, et al., (Jr. Clin. Invest., 63, pp 836-848 (1979) and Frasch,et al. (in The Pathogenic Neisseriae pp 633-640, edited by G. Schoolnik,Praeger, New York (1985)) have used hydrophobic complexing to make outermembrane protein-polysaccharide vaccines. However, these researchers didnot disclose the hydrophobic complexes disclosed herein.

[0014] Coon, et al (Journal of Immunology, Vol. 110, pp 183-190 (1973))found that when lauric acid was heavily conjugated covalently to a largeprotein, bovine serum albumin, humoral immunogenicity was absent, butcell mediated immunity could be induced. In this work, lauric acid wasnot used to enhance humoral immunogenicity and peptides or proteinfragments or hydrophobic complexes were not used.

[0015] Hopp disclosed (Molec. Immunol., Vol. 21, pp 13-16 (1984))addition of dipalmityl-lysine to a peptide to enhance itsimmunogenicity. The immunopotentiation reported by Hopp was exceedinglyshort-lived and induced peak titers that were only 5.1 fold greater thanhis control values. Furthermore, immunization as disclosed therein wascarried out using Freund's adjuvant, which is not acceptable for use inhumans.

[0016] Bessler, et al. (Immunobiology, Vol 170, p 239(1985)) reportedusing a tripalmitoyl pentapeptide analog of E. Coli lipoprotein as anadjuvant for sheep red blood cells, which were co-administered in amixture. These researchers also covalently linked the tripalmitoylpentapeptide to another peptide in order to boost the immune response.However, no enhancement of immunogenicity is evidenced therein.

[0017] Ballou (Science, Vol 228, pp 996-999(1985)) worked with naturallyoccurring antigens containing repeating epitopes such as those found inmalaria to genetically engineer a cloned portion of the organism having32 repeats of 4 amino acids (16 repeats of an 8 amino acid epitope withan additional 32 amino acid tetracycline-resistant marker peptide). Thissystem was found effective in small animals when used with alum orFreund's adjuvant. These investigators did not, however, enhanceimmunogenicity in accord with the teachings of this invention.

[0018] Audibert, et al., (Proceedings National Academy of Science, USA,Vol. 79, pp 5042-5046 (1982)) used glutaraldehyde to polymerize apeptide, but found that the peptide was not immunogenic unless both aprotein carrier such as bovine serum albumen or poly (LD-Ala)--ply(L-Lys) plus an adjuvant such as Freund's or muramyl dipeptide was used.

[0019] Liposomes have been considered as adjuvants. However, liposomesare entirely lipid and differ fundamentally from the system disclosedherein.

[0020] Morein and Simons (Vaccine, Vol. 3, pp 83-93 (1985)) describedimmunogenic complexes called iscoms between antigenic proteins andglycosides. The instant invention is fundamentally different from theconstructs of Morein and Simons, since the present invention does notrequire glycosides.

[0021] The production of immune response to P. falciparum, the causativeagent of malaria, is of particular concern. Worldwide, malaria is themost common serious infectious disease affecting humans. The P.falciparum has a tandemly repeated circumsporozoite (CS) tetrapeptide(NANP), which has been the subject of much vaccine research. G. N.Godson, in Molecular Approaches to Malaria Vaccines, discusses therepeated antigenic sequences in the circumsporozoite protein. When ananimal is injected with sporozoites most of the antibodies it raises aredirected against the CS protein, and specifically against the repeatingepitope thereof.

[0022] European patent application EPA 191,748 (which is incorporatedherein by reference) published Aug. 20, 1986 refers to an E. coliexpression vector having a coding sequence for all or a portion of therepeat unit of the protein CS and discloses a process for purifying theimmunogenic polypeptide from the E. coli culture.

[0023] European patent publication EPA 192,626, published Aug. 27, 1986refers to an immunogenic polypeptide capable of conferring immunity toinfection with P. Faciparum in mammals. The immunogenic polypeptidecomprises four or more tandem repeat units of the CS protein. The repeatunit is a tetrapeptide having the sequence Asn Ala Asn Pro. Both of theEPA application identified above are incorporated herein by reference.

[0024] PCT published application WO87/06939 published Nov. 19, 187teaches a process for isolating and purifying the CS protein expressedin recombinant E. Coli.

[0025] Dame, et al. discloses, in U.S. Pat. No. 4,707,357, ananti-malarial immunogenic stimulant comprising an immunogenic carrierand a peptide sequence of between two and 100 consecutive repeats of asequence Asn X Y Pro, wherein X is Ala or Val and Y is Asn or Asp. Thecarriers disclosed therein include soluble molecules such as proteinsand polysaccarides and particles such as liposomes and bacterial cellsor membranes thereof. The peptide is attached to the carrier by an amidebond formed between a carboxylic acid or amino group of a carrier and anamino or carboxylic acid group of the peptide. The bonding may bethrough either an ether or ester linkage. Groups such as terminaldiamines with one to 10 methylene carbons joining the amines are alsorecited as carriers. Preferred carriers disclosed are tetanus toxoid andamphoteric proteins having a lipophilic portion and a hydrophilicportion.

[0026] Patent publication WO86/05790, published Oct. 9, 1986, disclosesimmunogenic antigen-carrier protein conjugates for use as vaccinesagainst malaria. The conjugates contain the peptide H—(Asn Ala AsnPro)₃—OH, also designated (NANP)₃. This application also describes apreferred carrier such as tetanus toxoid. Other carriers includediphtheria toxoid and synthetic peptides and polymers comprising lysineand arginine groups. The peptide is coupled to the carrier usingglutaraldehyde as a coupling reagent or adding a cysteine residue to theN-terminal of the peptide and using another conventional ester as acoupling reagent.

[0027] Achlessinger, et al., U.S. Pat. No. 4,769,235 (which isincorporated herein by reference) refers to eptiopes having the sequenceof an immunodominant epitope from the repeat region of the CS proteinwhich is shorter in length than the repeating unit of the CS protein.This peptide was active as a vaccine when coupled with a conventionalcarrier.

[0028] Patent publication WO86/00911 published Feb. 13, 1986 refers tothe sue of a peptide an amino acid sequence Pro Asn Ala Asn repeated 23or more times and adsorbed or coupled to a conventional vaccine carrierprotein.

[0029] Alum absorbed vaccines containing various forms of the CS epitopehave not been sufficiently immunogenic for general human use. Manyprotein carriers and liposomes recited in the prior art documentsrequire lipid A or other adjuvants not acceptable for human use.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The instant invention provides an immunopotentiating system whichwill render peptides (including small peptides) immunogenic and whichwill enhance the immunostimulating properties of larger peptides,proteins, and protein fragments. Immunostimulating can be defined as thecapacity to induce a cytotoxic T cell lymphocyte response and/or anantibody response in a mammal. The desired amino acid sequences may bemade by synthesis of amino acid sequences and/or polymerization, byextraction from the pathogens, or by recombinant means. Peptides orproteins may be characterized by variations from the immunostimulatingsequences of the natural pathogens by addition, deletion, or insertionof other amino acids or by the attachment of additional sequences. Thepeptides may be positively or negatively charged or may be neutral. Thepeptides may be replicated to form tandem repeats.

[0031] The peptides and proteins used in the method of the invention maynaturally contain cysteine residues. However, the natural presence ofcysteine(s) is not required.

[0032] The hydrophobic foot which is attached to the immunostimulatingsequence may vary in structure. A preferred hydrophobic foot comprisesan aliphatic carbonyl group containing from 8 to 18 carbon atoms. In apreferred embodiment the group contains an alkanoyl moiety. Aparticularly preferred foot is lauroyl. Molecules of this type areeasily added to the amino terminus of a synthetic peptide while thepeptide is still on the resin used for synthesis. Peptides may besynthesized by the solid phase method described by Merrifield (Soc., Vol85, pp 21-49 (1963)). When a synthetic peptide is used, the alkanoyl,preferably as the chloride, can be reacted with the peptide on theresin.

[0033] The alkanoyl may also be added to the amino terminus by reactionof an alkanoyl acid such as lauric acid. To avoid side reactions, freeamino groups may be blocked to assure that the alkanoyl group isattached to the end of the peptide. It is also possible to attach thealkanoyl group on the carboxy terminal using lysine as thecarboxyterminal amino acid and reacting the alkanoyl with the epsilonamino group of the amino acid by conventional means.

[0034] The hydrophobic anchor may also be a hydrophobic peptide of about3 to 50 amino acids (though preferably ≦24 amino acids) in length. Inthe instance where the immunogen is a peptide synthesized by sequentialsolid or liquid phase synthesis, the peptide may be added to theterminus (either amino or carboxy). A preferred hydrophobic peptide is apentapeptide. The peptide Phe-Leu-Leu-Ala-Val is a preferred embodiment.The amino acids Tyr, Phe, Trp, Pro, Val, Ile and Leu are particularlyuseful in providing hydrophobicity. Hydrophobic amino acids of longerchain length can also serve the function of the hydrophobic foot so longas the length of the hydrophobic foot does not exceed about 24 aminoacids. The peptide should not be rendered totally water insoluble in thepresence of detergent.

[0035] When the peptides are synthesized, cysteine(s) may be addedduring the synthesis, of the peptide. Cysteine may also be added topreviously synthesized sequences by a carbodiimid reaction. The cysteineis useful for effecting dimerization or cyclization of the peptides.Unless reducing agents are present, dimerization occurs spontaneouslyfollowing deblocking and cleavage of the peptide when one cysteine ispresent in the peptide. In a preferred embodiment one cysteine islocated between the hydrophobic foot and the peptide epitope. When theconstruct contains two cysteines, cyclization is accomplishedspontaneously in dilute solution after de-blocking and cleavage of thepeptide. Ferricyanide oxidation of the peptide in the dilute solutioncauses formation intrachain (but not inter-chain) disulfide bonds.

[0036] In an embodiment of the invention, one cysteine residue is addedto provide for dimerization of both the hydrophobic foot and thehydrophilic epitope. Dimerization appears to provide more stable bindingto the proteosome by providing two hydrophobic feet for the epitope. Thedimerized constructs also provide for more stable interaction with theantigen. The cysteine may be placed at either the carboxy or aminoterminus of the epitope.

[0037] When the immunostimulating epitope is produced by geneticengineering means, the nucleotide sequence required for production of apeptide which is the hydrophobic foot and any desired cysteine(s) may beattached to the nucleotide giving rise to the immunostimulating epitope.

Materials and Methods

[0038] Proteosome preparation:

[0039] Proteosomes were prepared from Group B type 2b meningococci.Proteosome preparation consisted of two stages. The first stage was doneby either of two methods: (1) isolation of meningococcal outer membranevesicles by extraction from an aqueous suspension of whole meningococcias previously described (Zollinger et al., J. Clin. Invest., 63, page836-848, 1979) or (2) collection of a direct bacterial cell extractprecipitate. The direct cell extracts were obtained by extraction ofpacked bacterial cells for one hour at room temperature with one literper 100 grams of cells of a solution containing 0.1 M sodium acetate pH5.0, 0.5 M CaCl₂ and 3% Empigen BB. Ethanol was added to the mixture toa concentration of 20% v/v and the precipitate removed by centrifugationat 16,000× g for 10 minutes. Additional ethanol was added to thesupernatant to a final concentration of 45% and the precipitate,constituting the direct cell extract, was collected by centrifugation.

[0040] The second stage of the proteosome preparation consisted ofisolating the outer membrane proteins from the other membrane componentsby dissolving either of the products from the first stage (i.e. eitherthe vesicles or the direct cell extract) at a concentration ofapproximately 2 mg protein/ml in a buffer (hereafter referred to asTEEN-1%) containing 0.05 molar trishydrochloride (hydroxyacetyl aminomethane), 0.15 M NaCl, 0.01 M EDTA (ethylene diamine tetra-acetate) and1% Empigen BB (Albricht and Wilson, Cumbria, England) brought to pH 8.0.The proteins were then precipitated three times by addition of solidammonium sulfate at 500 g/l of protein solution. The precipitates werecollected by centrifugation at 30,000× g for 20 minutes and redissolvedat about 2 mg protein/ml in TEEN-1%. The final precipitate was dissolvedwith the aid of a water bath sonicator at about 2 mg/ml, centrifuged at16,000× g for 20 minutes to remove insoluble material and then dialyzedagainst TEEN-0.1% to remove any residual ammonium sulfate. (The finalconcentration of Emigen BB can be 0.1% to 1.0%). Products are stored at−20° C. (or, for short periods at 4° C.).

[0041] The proteosomes prepared from bacteria other than those preparedfrom meningococci may also be prepared and used by the same methodology.

[0042] In certain instances, the hydrophobic foot attached through thecysteine may be sufficient to provide needed antigenicity without use ofproteosomes.

[0043] Epitope replication:

[0044] The peptide may be synthesized as a repeating unit wherein thesequences are in tandem as many times as synthesis will allow.Replicates of two to six times have been used with increasinglyenhancing effects. Epitope replication enhances the immunogenicity ofthe peptide epitope. When the repeating units are complexed with ahydrophobic foot prepared with the methodology described below, atotally non-immunogenic peptide can be made immunogenic without addedadjuvants and even without the proteosomes. Complexing the replicatedpeptides directly with proteosomes is also effective. When the cysteineis present with the replicated epitopes and the proteosomes, the systemis optimal. Because complexing is dependent upon hydrophobic sites, thenumber of peptide molecules that can be complexed to the proteosome canbe far greater than can be complexed by ordinary covalent bondingsystems. When complexed with protein, 6-30 protein molecules mayassociate with each proteosome.

[0045] Any vaccine may be made by the method of the invention, includingvaccines against parasitic, viral, bacterial, and fungal infection.Vaccines to prevent pathological response to toxic chemical andbiological agents and against malignancies as well as vaccines toprotect from pregnancy may be made by methods of the invention.

[0046] Preparation of immunogenic peptide vaccines:

[0047] Either of two complexing methods may be used, 1) Dialysis or 2)Lyophilization:

[0048] 1. Dialysis:

[0049] a. Combine components in TEEN-1%: The proteosomes, stored inTEEN-1% buffer (see above) at a concentration ≧1 mg/ml (usually 1.5-2.5mg/ml), are added to a TEEN-1% solution of the peptide with thehydrophobic foot (and the cysteine &/or the replicated epitope asdesired) in a beaker or test tube. Ratios of protein:peptide(weight:weight) that have been used have ranged from 1:1 to 1:40. Theusual ratio has been 1:1 although, depending on the circumstances, 1:4or higher may be preferable. Note that the concentration of the peptidein the solution prior to combination with proteosomes must be highenough so that the concentration of both the peptide and the protein inthe combined mixture is ≧1 mg/ml when the components are at equal ratiosand, when the ratio is not 1:1, the concentration of the lessconcentrated component is ≧0.50 mg/ml and preferably, ≧0.75 mg/ml. Forexample, if the proteosomes are at 1.1 mg/ml, the peptide must be at 10mg/ml prior to combining at a 1:1 ratio. While these minimalconcentrations are not absolute and although successful vaccines havebeen made using protein concentrations that are more dilute (when thepeptide:protein ratio is significantly ≧1:1) the method suggested hereis more consistently successful.

[0050] b. Dialyze: The mixture was transferred to dialysis bags that,due to their low molecular weight cutoff, retain both the peptide andthe protein while allowing the detergent (usually Empigen-BB) in theTEEN-1% to dialyze away. For this reason, Spectra-Por 6 (or 7) dialysistubing with molecular weight cutoff of 1000 are routinely used to becertain that as much peptide as possible is retained for complexing tothe proteosomes. The dialysis tubing (closed using special spectra-porclosures) was washed just prior to use with pyrogen-free distilled waterand then Phosphate Buffered Saline pH 8.5 (PBS-8.5). This latter bufferis the buffer against which the proteosome-peptide mixture wasexhaustively dialyzed (e.g. at a ratio of 200-250:1 for 10 days changingthe dialysis fluid daily) and consists of 0.025 M Na₂HPO₄ plus 0.15 MNaCl (normal saline). On the last day of dialysis, the buffer is changedto standard Phosphate buffered saline, PBS (Na₂HPO₄+NaH₂PO₄+NaCl at pH7.4). Under certain circumstances, dialysis may be able to be shortenede.g. to 5 days with 2 changes of fluid per day.

[0051] c. Collect vaccine: Solution was collected from dialysis bag(s).Dialysis bags were washed with 20% of their volume with PBS and the risewas combined with vaccine. The vaccine was filtered through a 0.22 μmfilter (the vaccine may need to be pre-filtered through a 0.8 or 0.45 μmfilter) or just a 0.45 μm filter. The protein content was measured (e.g.by optical density at 280 nm or Lowry assay), and samples were taken foramino acid analysis, HPLC and other analyses, The samples were thendiluted with PBS to result in a 0.4 mg protein per ml solution. Thefinal vaccine was then diluted 1:1 with either Normal Saline (with 0.02%merthiolate) to result in a 0.2 mg/ml solution which is administered at0.5 ml per intramuscular dose. Alternatively, if desired, the vaccinecan be adsorbed to alum by diluting 1:1 with a solution of alum insteadof Normal Saline, allowing to sit at room temperature for 2 hrs. withoccasional stirring and then at 4° C. for 3-18 hrs. It should beemphasized that the data indicate that the vaccine works perfectly wellwithout alum and that the only reason for adding alum is to evaluate itsrole in the long term human response.

[0052] 2. Lyophilization:

[0053] Instead of combining the proteosomes and peptide (with ahydrophobic foot) in TEEN-1%, these components may be immunogenic whencomplexed by simply lyophilizing them together according to thefollowing procedure:

[0054] Proteosomes are removed from TEEN-1% by precipitating them byadding three volumes of 100% ethanol to one volume of the proteosomes,allowing to stand at 4° C. for one hour and then centrifuging them at800-1000 g for 15 minutes; washing the proteosomes three times by addingthe same amount of 100% ethanol as previously used and re-centrifugingas before, then resuspending the proteosomes in PBS to a concentrationof 2 mg/ml.

[0055] The peptide (with its hydrophobic foot and, if desired, cysteineand replicated epitopes) is then redissolved in PBS at 2 mg/ml (oranother concentration as described above if a peptide:protein ratio >1:1is desired). The dissolved peptide is added to the proteosome suspensionand mixed. The mixutre is lyophilized and, following lyophilization,resuspended to 1 mg protein/ml using distilled water. The product isthen filtered, analyzed, diluted and added to saline or alum asdescribed above.

[0056] Addition of Cysteine:

[0057] Adding the amino acid cysteine is employed in this system ineither of two ways:

[0058] a. Dimerization: Adding one cysteine provides for dimerization ofboth the hydrophobic foot and the hydrophilic epitope. This componenthas been shown to be effective in enhancing the immunogenicity of apeptide in either of 3 ways: i) in conjunction with component thehydrophobic foot plus the proteosomes, ii) in conjunction with epitopereplication, or iii) in conjunction with a hydrophobic foot and anepitope replication.

[0059] Dimerization provides two hydrophobic feet for the epitope, toprovide more stable binding to the proteosomes or effect formation ofauto-micelles. Furthermore, the two epitopes provided for by thedimerization can yield a more stable interaction with antigenrecognition cells and may improve conformation of the peptide epitope.

[0060] b. Cyclization: Two cysteines are added to either end of thehydrophilic peptide epitope (i.e one cysteine between the epitope andthe hydrophobic foot and one cysteine at the other end of thehydrophilic epitope). After the hydrophobic foot has been added thepeptide may be deblocked and the peptide cyclized using an oxidizingagent such as ferricyanide.

[0061] Epitope replication:

[0062] Epitopes may be repeated in tandem as many times as synthesiswill allow. Replication enhances the immunogenicity of the peptideepitope. When prepared with the methodology described below, a totallynon-immunogenic peptide can be made immunogenic without added adjuvantsand even without the proteosomes. Complexing the replicated peptideswith proteosomes is also effective. Epitope replication may be used inconjunction with addition of cysteine for addition to a hydrophobic footand/or proteosome.

[0063] The components of the inventive constructs can by complexed byany means known in the art. Any synthetic or cloned peptide can have ahydrophobic foot and cysteine added and therefore any peptide can bemade immunogenic by this system which differs from chemical covalentcomplexing which depends on the correct chemistry to attach and orientthe peptide epitope appropriately.

[0064] Proteosomes, in their native hydrophobic state, have speciallymphocyte activating properties which allow them to act as both aprotein carrier and an adjuvant. Since they are not chemically modified,but retain their multimolecular hydrophobic and membranous structure inthe vaccine, their ability to immunopotentiate the immunogenicity of thepeptides complexed to them is due to these special properties which areretained by the methods outlined above.

[0065] When using the hydrophobic foot with the cysteine and thereplicate epitopes, peptide immunogenicity can be obtained even withoutaddition of proteosomes. Toxicity and side effects would minimal.

EXAMPLES

[0066] The amino acid sequences of some of the peptides used to produceand operate this invention are given in TABLE 1. PepG is an example of acyclic peptide—it has two cysteines which have been joined in adisulfide bond to make a cyclic loop in the peptide. PepM1 isnon-cyclic, is without an added cysteine and contains the native epitopeonly once. PepCM1 has an added cysteine at the amino terminus as doespepCM3 and pepCM5. PepCM3 has three replicates of the native M epitopeand pepM5 and pepCM5 have five such replicates. PepL1 has an epitope ofonly seven amino acids as does its cysteine-containing counterpart, CL1.

[0067] The data of tests done to produce and operate the invention aredetailed in TABLES 2-4. All vaccines were prepared as described below.Briefly, the peptides, with or without added cysteines, were synthesizedby standard solid phase technology. While still on the resin, a lauroylgroup was added to the amino terminus as described below or thepentapeptide hydrophobic foot, Phe Leu Leu Ala Val (FLLAV), was added bysimply continuing the synthesis. Except when noted otherwise, allvaccines were prepared by dissolving the peptides and/or the proteosomesin TEEN-1% detergent buffer and then exhaustively dialyzing away thedetergent.

[0068] As shown in TABLE 2, both normal mice (BALB/c) and mice that arenon-responsive to the adjuvant effects of lipopolysaccharide (C3H/HeJ),when immunized with either pepG alone, pepG in Freund's adjuvant, pepGwith proteosomes (but without any hydrophobic foot), or eitherlauroyl-pepG or FLLAV-pepG without proteosomes were totallynon-immunogenic (group 1, controls a-e). In marked contrast, pepG wasmade highly immunogenic by complexing it to proteosomes via either alauroyl hydrophobic foot (groups 2 and 4) or via the pentapeptidehydrophobic foot (groups 3 and 5). This was demonstrated in both BALB/cmice (groups 2 and 3) and C3H/HeJ mice (groups 4 and 5).

[0069] Similarly, pepCL1 which is non-cyclic, was made immunogenic inboth normal mice (group 15) and LPS non-responder mice (group 16). Asexpected, pepCL1 control groups were non-immunogenic (group 13, controlsa-d).

[0070] The M1 epitope was tested for immunogenicity in the system bothwith an added cysteine (groups 9-12) and without the cysteine (groups6-8). The cysteine was shown to be exceedingly important. Highimmunogenicity resulted from immunizing with either the standard 40 μgdose (group 11) or a sub-standard (8 μg) dose (group 12) of pepCM1complexed to proteosomes. This peptide, lauroyl-pepCM1, was mildlyimmunogenic (after three immunizations) without proteosomes (group 10).In contrast, pepM1, lacking the cysteine, exhibited only the mostminimal immunogenicity even with proteosomes (groups 7 and 8). Thecysteine was considered to be important because its free sulfhydrylgroup causes dimerization of both the epitope and the hydrophobic foot.Dimerization of the epitope may allow better recognition by antigenprocessing cells whereas dimerization of the hydrophobic foot promotesbetter complexing to proteosomes.

[0071] The role of replicated epitopes in promoting immunogenicity inthe system is detailed in TABLE 3. Once again, the peptides alone, evenwhen lengthened by replicating the epitope three times, werenon-immunogenic (group 17, control groups a-c). Nevertheless,immunization of normal BALB/c mice with the fatty acyl hydrophobic footvariant of this peptide, Lauroyl-pepCM3, resulted in high immunogenicityeven without proteosomes when the vaccine was prepared by the standarddialysis method (group 18). Note that Lauroyl-pepCM3 was non-immunogenicwhen not dialyzed (group 19). It was found that dissolving thelipopeptide in detergent and then dialyzing away the detergent iseffective for promoting formation of auto-micelles during the dialysis.

[0072] When Lauroyl-pepCM3 was complexed to proteosomes, immunogenicitywas even further enhanced (group 20). This vaccine was also effective inC3H/HeJ mice again demonstrating that the ability of the proteosomes toenhance immunogenicity was not due to the <1% contaminating LPS in theproteosome preparation (group 24). It is believed that the lack ofeffect from the Lauroyl-pepCM3 in C3H/HeJ mice (group 23) was due togenetic restriction of recognition of the M1 epitope in these mice (andnot insensitivity to the adjuvanticity of LPS) since a) Lauroyl-pepCM3does not contain LPS and b) complexing the peptide to proteosomes whichwere able to provide carrier-like T-cell influence resulted in animmunogenic vaccine (group 24).

[0073] The optimal nature of the system when each of the four components(the proteosomes, hydrophobic foot, the cysteine and the replicatedepitopes) were present was also demonstrated using pepCM3 with the FLLAVpentapeptide hydrophobic foot. Thus, FLLAV-pepCM3 was not immunogenicalone (group 21) whereas FLLAV-pepCM3 complexed to proteosomes was amongthe most immunogenic of all vaccines using the M1 epitope (group 22).

[0074] The role of the cysteine was also confirmed in conjunction withthe replicated epitope and the lauroyl hydrophobic foot. The pepM5control groups were non-immunogenic (group 25) as were the pepCM5control groups (group 28). But when pepM5 was supplemented with thelauroyl hydrophobic foot (group 26) or both the lauroyl foot andproteosomes (group 27) only mild immunogenicity ensued even though thepeptide was 47 amino acids long and had the M epitope repeated fivetimes. In marked contrast, the Lauroyl-pepCM5 (which contains the addedcysteine) was highly immunogenic (group 29) and complexingLauroyl-pepCM5 to proteosomes further enhanced the immunogenicity (group31) to maximal levels. When pepCM3 was incubated without dialysis,immunogenicity was markedly reduced (group 30). In C3H/HeJ mice,Lauroyl-pepCM5 was only minimally immunogenic (group 32) butProteosome-Lauroyl-pepCM5 was clearly immunogenic (group 33). This datais consistent with the previous data obtained in C3H/HeJ mice asdescribed above.

[0075] As shown in TABLE 4, effective proteosome-hydrophobic footvaccines can also be made without using the dialysis method (describedin the Methods section). Although the dialysis method appears to beoptimal (groups 36-40), excellent immunogenicity can also be obtained bylyophilization of a saline or water mixture of the peptide (containing ahydrophobic foot, e.g. Lauroyl-CM1) with proteosomes that havepreviously been removed from the empigen detergent (group 35). Simplymixing the components together in saline, is not as effective as eitherlyophilization or dialysis although significant immunogenicity isattained this way (group 34). There may be applications in which thealternate methodologies described would be advantageous.

[0076] Also shown in TABLE 4 is the effect of varying the ratio ofproteosome to peptide in the vaccine from 1:1 to 1:16 (groups 36-40). Asis clearly evident, each of the vaccines was highly immunogenic. Theimplications of being able to use a ratio with more peptide per unit ofprotein are a) less protein needs to administered in order to generatean effective immune response so that the possibility of side effectsfrom the protein can be diminished and b) if a maximum amount of proteinis administered, the amount of peptide that can be given iscorrespondingly increased. This increase in the amount of peptide thatcan be given may be critical to the development of a successful vaccinewhen the peptide epitope is particularly refractory to potentiation ofimmunogenicity. TABLE 1 AMINO ACID SEQUENCES OF SEVERAL TRYPANOSOMALPEPTIDES TESTED IN PROTEOSOME-HYDROPHOBIC FOOT VACCINE SYSTEM No. CODESEQUENCE 1 pepG

2 pepM1 YG(VPVAQTQTG) (Seq. No. 4) 3 pepCM1 CYG(VPVAQTQTG) (Seq. No. 5)4 pepCM3 CYG(VPVAQTQTG)₃ (Seq. No. 6) 5 pepM5 YG(VPVAQTQTG)₅ (Seq. No.7) 6 pepCM5 CYG(VPVAQTQTG)₅ (Seq. No. 8) 7 pepL1 (KYNATKA) (Seq. No. 9)8 pepCL1 C(KYNATKA) (Seq. No. 10)

[0077] The sequences within the parentheses are homologous with thesequences of the peptides in the native organism.

[0078] pepG=Tyr Gly Gly (Gly Cys Thr Gln Ile Thr Glu Pro Thr Cys Asn S=S

[0079] pepM1=Tyr Gly (VaLl Pro Val Ala Gln Thr Gln Thr Gly)

[0080] pepCM1=Cys Tyr Gly (Val Pro Val Ala Gln Thr Gln Thr Gly)

[0081] pepCM3=Cys Tyr Gly (Val Pro Val Ala Gln Thr Gln Thr Gly)₃

[0082] pepM5=Tyr Gly (Val Pro Val Ala Gln Thr Gln Thr Gly)₅

[0083] pepL1=Lys Tyr Asn Ala Thr Lys Ala

[0084] PepCl1=Cys Lys Tyr Asn Ala Thr Lys Ala TABLE 2 ANTI-PEPTIDEANTIBODY TITERS IN SERA OF MICE AFTER PRIMARY (1°), SECONDARY (2°) ANDTERTIARY (3°) IMMUNIZATION WITH PEPTIDES WITH LAUROYL OR FLLAVHYDROPHOBIC FEET &/OR CYSTEINE &/OR PROTEOSOMES ANTI-PEPTIDE SERUMANITBODY GRP MOUSE TITERS POST IMMUNIZATION§ NO. STRAIN§ VACCINE123° 1B, J pepG Controls (a-e)* <50 <50 <50 2 B Proteosome-Lauroyl-pepG 400204,800 204,800 3 B Proteosome-FLLAV-pepG 400 12,800 102,400 4 JProteosome-Lauroyl-pepG 200 6,400 51,200 5 J Proteosome-FLLAV-pepG 100102,400 409,600 6 B pepM1 Controls (a-c)* <50 <50 <50 7 B Lauroyl-pepM1<50 200 400 8 B Proteosome-Lauroyl-pepM1 <50 400 400 9 B pepCM1 Controls(a-c)* <50 <50 <50 10 B Lauroyl-pepCM1 <50 <50 3,200 11 BProteosome-Lauroyl-pepcM1 400 102,400 409,600 12 BProteosome-Lauroyl-pepCM1(8 ug) 200 102,400 204,800 13 B, J pepCL1Controls (a-d)* <50 <50 <50 14 B Lauroyl-pepCL1 800 400 800 15 BProteosome-Lauroyl-pepCL1 50 200 51,200 16 J Proteosome-Lauroyl-pepCL150 400 51,200 # 1:50.

[0085] TABLE 3 ANTI-PEPTIDE ANTIBODY TITERS IN SERA OF MICE AFTERPRIMARY (1°), SECONDARY (2°) AND TERTIARY (3°) IMMUNIZATIONS WITHPEPTIDES WITH LAUROYL OR FLLAV HYDROPHOBIC FEET AND/OR CYSTEINES AND/ORREPLICATED EPITOPES AND/OR PROTEOSOMES ANTI-PEPTIDE SERUM ANTIBODY GRPMOUSE TITERS POST INMUNIZATION§ NO. STRAIN§ VACCINE 1° 2° 3° 17 B, JpepCM3 Control groups (a-c)* <50 <50 <50 18 B Lauroyl-pepCM3 400 102,400102,400 19 B Lauroyl-pepCM3 (non-dialyzed) <50 <50 100 20 BProteosome-Lauroyl-pepCM3 6,400 102,400 409,600 21 B FLLAV-pepCM3 <50 5050 22 B Proteosome-FLLAV-pepCM3 <50 204,800 6,553,600 23 JLauroyl-pepCM3 <50 50 50 24 J Proteosome-Lauroyl-pepCM3 <50 800 204,80025 B pepM5 Control groups (a-c)* <50 <50 <50 26 B Lauroyl-pepM5 200 40012,800 27 B Proteosome-Lauroyl-pepM5 200 1600 12,800 28 B, J pepCM5Control groups (a-c)* <50 <50 <50 29 B Lauroyl-pepCM5 800 204,800204,800 30 B Lauroyl-pepCM5 (non-dialyzed) 100 12,800 25,600 31 BProteosome-Lauroyl-pepCM5 400 25,600 3,276,800 32 J Lauroyl-pepCM5 50100 100 33 J Proteosome-Lauroyl-pepCM5 200 25,600 51,200

[0086] TABLE 4 EFFECTS OF THE COMPLEXING METHOD AND THEPROTEOSOME:PEPTIDE RATIO ON THE ABILITY OF PROTEOSOMES TO ENHANCE THEIMMUNOGENICITY OF PEPTIDE LAUROYL-CM1 ANTI-PEPTIDE SERUM ANTIBODY GRPCOMPLEX PROTEOSOME:PEPTIDE TITERS IMMUNIZATION§ No. METHOD* RATIO POST123° 34 Mix 1:1 400 6,400 51,200 35 Lyophilize 1:1 800 12,800 409,600 36Dialyze 1:1 12,800 409,600 6,553,600 37 Dialyze 1:2 25,600 819,200819,200 38 Dialyze 1:4 6,400 819,200 1,638,400 39 Dialyze 1:8 12,800819,200 1,638,400 40 Dialyze  1:16 51,200 1,638,400 3,276,800 # o.d.units & b) twice the value of prevaccination sera diluted 1:50.

[0087] TABLE 5 ANTI-MENINGOCOCCAL IgG ANTIBODIES IN SERA OF MICEIMMUNIZED AND BOOSTED WITH PROTEOSOME-HYDROPHOBIC FOOT-PEPTIDE VACCINESUSING EITHER THE LAUROYL OR THE FLLAV HYDROPHOBIC FOOT GRPANTI-MENINGOCOCCAL IgG NO VACCINE ANTIBODY TITERS § 1 Controls <50 2Proteosome-Lauroyl-pepG 102,400 3 Proteosome-FLLAV-pepG 409,600 # sera,obtained 2-3 weeks after each immunization, were tested in an ELISA forIgG antibodies against meningococcal outer membrane # proteins. Titersshown are the highest serum dilutions obtained after two or threeimmunizations which had ELISA values that # were a) more than 0.1 o.d.units & b) twice the value of pre-vaccination sera diluted 1:50.

[0088] Longer peptides and proteins may also be potentiated by methodsof the invention. Many extracted or cloned polypeptides (especiallytransmambrane polypeptides) naturally have hydrophobic ancors which arefrequently 15 to 30 amino acids long. The immunogenicity of suchpolypeptides may also be enhanced by extending the native hydrophobicanchor or by adding another hydrophobic anchor according the methods ofthe invention. A preferred decapeptide,Gly-Gly-Tyr-Cys-Phe-Val-Ala-Leu-Leu-Phe is a preferred embodimentbecause of appropriate size and composition to allow for easypurification of a recombinant anchored protein. Native sequences can beof such length and composition as to hinder extraction and purification.

[0089] The hydrophobic anchor sequence is preferably added to thecarboxy-terminus of the selected recombinant protein by geneticengineering methods. Hence, the polynucleotide that encodes the anchorcan be added to the 3′ end of the gene that encodes for the desiredrecombinant protein. Alternatively, the polynucleotide that encodes theanchor may also be added to the 5′ end of the selected protein. Asanother embodiment, the polynucleotide that encodes the anchor may beadded to both the 5′ and 3′ termini of the sequence that encodes theselected protein. For conventional techniques to accomplish constructionof these vectors, see T. Maniatis, et all, Molecular Cloning (ALaboratory Manual), Cold Spring Harbor Laboratory (1982). The constructscan be complexed to the proteosomes by dialysis or lyophilization asdescribed above in methods for preparation with peptides. Similarly, thehydrophobic foot may be attached by the methods indicated for attachmentto peptides as an alternative to production in a recombinant molecule asdescribed above.

[0090] Ratios of proteosomes to anchored recombinant protein(weight:weight) ranges from 1:1 to 1:20. Preferred ratios are between 1:and 1:3 for polypeptides or proteins.

[0091] Since hydrophobic complexing is more physical than chemical, andsince hydrophilic protein epitopes are always left conserved, exposed,and unaltered, antibodies generated against the epitope will easilyrecognize the protein or epitope therein and will, therefore, befunctional against the pathogen from which the epitope is derived.

[0092] Vaccine compositions of the invention may be introduced into thepatient by conventional means, including parenteral routes (for example,subcutaneous, intradermal, intramuscular) and by direct application tomucous membranes. Lyophilized compositions may be “snorted” into thenasal cavity. Dosage will depend on the particular agent administered.

[0093] An example of the value of the method of the invention isillustrated by use with a recombinant protein (R32RL), a 384 base pairfragment encoding 32 tetrapeptide repeats [(Asn Ala Asn Pro)₁₅₋ (Asn ValAsp Pro)]₂ of the P. falciparum CS protein, rendered immunogenic byadding the hydrophobic foot, cysteine-containing decapeptide anchor toits carboxy terminus to create R32Ft. R32Ft is immunogenic in vaccinetesting when used alone and such immunogenicity is markedly enhancedwhen it is complexed to proteosomes via the added hydrophobicdecapeptide anchor described above. (It should be understood that theexamples provided herein are illustrative only and do not limit thescope of the present invention to the specific vaccine components northe particular recombinant protein used therein.

[0094] Construction of the Anchored Recombinant Protein. R32Ft:

[0095] Ten micrograms of expression vector pAS1 (ATCC 39262, more fullydescribed in U.S. Pat. No. 4,578,355, which is incorporated herein byreference) was digested with restriciton endonuclease BamHI (25 units)in 200 μl medium buffer [comprising 50 mM Tris, 5 mM NaCl, 1 mMdithiothreitol (DTT), and 10 mM MgCl, having a pH of 7.5] for 1.5 hoursat 37° C. One hundred nanograms of the BamHI-cut pAS1 was ligated with20 ng of a synthetic linker having the following sequence:5′-GATCCCGGGTGACTGACTGA    -3′ (SEQ. NO. 14)3′-    GGCCCACTGACTGACTCTAG-5′

[0096] The resulting plasmid, pT17, was identified with one linkerinserted into the BamHI site of pAS1. This vector retains the BamHIsite, introduces a uniique SmaIsite, and results in the insertion of TGAtermination codons in all three reading frames downstream of the ATGinitiation codon of the cII ribosome binding site.

[0097] Fourth micrograms of purified pUC8 clone 1, a pUC8 clone [Viera,et al., Gene, 19:259 (1982)] containing the CS protein coding sequenceas a 2337 base pair EcoRI fragment of gamma-mPF1 inserted into the EcoRIsite of pUC8 [Dame et a;., Science 225:593 (1984)] was digested withrestriction endonuclease XhoII in 400 μl of medium buffer for 1.5 hoursat 37° C. The resulting 192 base pair fragment, encoding 16 tetrapeptiderepeats [(asn-Al-Asn-Pro)₁₅-(Asn-Val-Asp-Pro)]₂ of the P. falciparum CSprotein, was isolated by electrophoresis on a 5% polyacrylamide gel(PAGE) and recovered by electroelution.

[0098] Expression vector pT17 (10 μg) was digested with restrictionendonuclease BamHI (25 units) in 200 μl medium buffer (described above)for 1.5 hours at 37° C. The Xho II CS protein gene fragment (1 230 g)was then ligated into this vector (100 ng) in 30 μl ligase buffer(comprising 50 mM Tris, 1 mM DTT, 10 mM MgCl₂, and 100 μM rATP, havingpH of 7.5) with one unit of T4-DNA ligase for 16 hours at 4° C.

[0099] The ligation mixture was transformed into E. coli strain MM294CI+[Smithkline French]. Ampicillin resisitant colonies were obtained andscreened for insertion of the Xho II gene fragment into pT17. A plasmidwith the correct construction, pR16, was identified and transformed intoE. coli strain MM294CI+.

[0100] Expression vector pR16 was digested with restricitionendonuclease BamHI as described above and a second Xho II CS proteingene fragment ligated into the vector. The ligation mixture wastransformed into E. coli strain MM294CI+, ampicillin resistant coloniesthereof selected and a plasmid with the correct construction, pR32,containing 32 repeats of the CS tetrapeptide, identified and transformedinto E. coli strain MM294CI+.

[0101] Expression vector pR32 (10 μg) was digested by restrictionendonuceases SmaI and SalI in 200 μl medium buffer (described above) for1.5 hours at 37°.

[0102] C. The synthetic DNA hydrophobic decapeptide anchor sequence (1μg) identified below was then added and ligated to the SmaI/SalI cutpR32 (100 ng) in 30 μl ligase buffer with one unit of T4-DNA ligase at4° C. for 16 hours. The hydrophobic decapeptide sequence was 5′ GGT GGTTAC TGC TTC GTT GCT CTG CTG TTC TGA G (SEQ. NO. 12) 3′ CCA CCA ATG ACGAAG CAA CGA GAC GAC AAG ACT CAGCT

[0103] The ligation mixture was transformed into E. coli strainMM294CI+. Ampicillin resistant colonies were obtained and screened forthe insertion of the decapeptide into pR32. A plasmid with the correctconstruction, pR32Ft, was identified and transformed into E. coli strainAR58 (CI⁸⁵⁷) and tested for expression of the gene product.

[0104] Cells were grown in Luria-Bertani Broth (LB) at 32° C. to anabsorbance of 650 nm (A₆₅₀) of 0.6 and temperture induced at 42° C. for3 hours to turn on transcription of the PL promoter of the expressionplasmid and subsequent translation of the CS protein derivative. Cellswere sampled in 1 ml aliquots, pelleted, resuspended in lysis buffer(comprising 10 mM Tris-HCl, 25% (vol/vol) glycerol, 2%2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and 0,1% bromophenolblue, having a pH of 7.8) and incubated in a 105° C. heating block for 5minutes. Proteins were separated by SDS-PAGE (12% acrylamide, 30:0.8acrylamide:bisacrylamide ratio).

[0105] Protein produced from E. coli was detected by Western Blotanalysis as described below in Example 2.

[0106] Purifiecation of R32Ft

[0107] The R32Ft peptide was purified from the expression system of theprevious example as disclosed below. All operations were performed onice unless stated otherwise.

[0108] Three 20-g E coli frozen pellets [SmithKline Laboratories] werecombined and thawed by suspending inot 240 ml of 50 mM Tris [Bio-Rad], 2mM ethylenediamine tetraacetic acid (EDTA) [Signma], 5% glycerol [Sigma]at pH 8.0 and stirring for one hour. Grade I lysozyme (48 mg, finalconcentration 0.2 mg/ml) and phenylmethyl sulfonyl floride (PMSF)[Signma], 1 ml at a concentration of 34 mg/ml in absolute ethanol wereadded and the suspension stirred for 30 minutes. The lysate was blendedfor 1 one-minute intervals in a blender and sonicated for 3 one-minuteintervals (Artek, model 300, medium probe). Sodium deoxycholate (DOC)[Sigma] was added to a final concentration of 0.1% (w/v). The suspensionwas stirred for 30 minutes, then centrifuged for 1 hour at 12000× g.

[0109] The supernatant was heated in a boiling water bath for 5 minuteswith stirring, cooled for one hour at ambient temperature, and thencentrifuged at 12000× g. Crude antigen was precipitated in a 10% to 40%ammonium sulfate peellet. The pellet was resuspended in 25 ml phosphatebuffered saline (PBS) and dialyzed extensively against PBS (Spectroportubing, NW cutoff 3000).

[0110] The sample was acidified to pH 2.0 by dropwise addition of 10%trifluoroacetic acid (TFA), stirred for 1 hour and centrifuged for 30minutes at 12000× g. The supernatant was collected and dialyzed into 10%PBS and lyophilized to rduce the volume to 5 ml. The solution wasrecentrifuged to clarify.

[0111] Final purification was carried out by high performance liquidchromatograph (HPLC) using a Waters system, including tow model 510pumps, and model 481 detector, automated gradient conntroller and an LKBmodel 2212 Helirac fraction collector with a semi-prep C-3 reverse phasecolumn. Protein elution was monitored at 214 nM. Buffer A was 0.05%TFA/water and Buffer B was 0.05% TFA in 90% MeCN/water. Flow was 9.5ml/min. The gradient started at 70% A, proceeded linearly to 50% A in 20minutes and was washed with 70% B for 8 minutes.

[0112] Proteins were neutralized by collection into equal volumes ofsaturated ammonium bicarbonate and assayed using a quick ELISA system.Protein peaks with strong ELISA activity were lyophilized andcharacterized by Western blot and amino acid analysis. Two peaks withactivity were eluted consistently at 45% and 48% B. The proteins wereindistinguishable by amino acid analysis and Western blot. Bothexhibited a single band migrating at 54 kd. Amino acid analsysis wasidentical.

[0113] The anchored recombinant proteins were complexed to theproteosomes via dialysis. Proteosomes in a concentration of 0.5-2.5mg/ml were added to solution of the recombinant protein with thehydrophobic foot to provide ratios of proteosomes to anchoredrecombinant protein (w/w) range of 1:1 to 1:20. The material wasdialized in accord with the teachings above.

[0114] Animal immunizations:

[0115] Groups of mice were dosed with 50 μg proteosomes with 50-100 μgR32Ft or with 50-100 μg R32Ft without the proteosomes. Adll injectionswere administered using saline as the carrier. No additional adjuvantswere used. Analysis of pooled sera from the groups of mice showed thatwhile the recombinant R32Ft alone was effective, the recombinant R32Ftcomplexed with the proteosome was at least 16 fold as effective as avaccine. Both C57B1 strain and BALB/c mice responded to the vaccines.When the animals were given booster shots (up to two boosters given) theimproved immune response was seen in all instances.

[0116] Individual rabbits were dosed with 100-200 μg R32Ft (recombinant)alone or complexed to 100 μg/dose of proteosomes. The recombinant R32Ftwith the proteosomes was about 10 fold as effective as the R32Ft havinghydrophobic foot but no proteosome complexed thereto.

[0117] Proteosomes can also be complexed with the anchored recombinantprotein by lyophilization in accord with the methods taught above.

[0118] Previous attempts to immunize mice with the protein R32LR(without the hydrophobic sequence) showed that protein to benon-immunogenic or, if given with complete freunds adjuvant or alum, tobe only poorly immunogenic.

[0119] gp160 vaccine against AIDS:

[0120] Proteosomes were constructed as indicated above and were storedat −70° C. in small aliquotes at concentration of >5 mg/ml (usually 6-7mg/ml) in TEEN buffer containing 0.1% (or, on occasion, 1%) Empigen BBdetergent. The proteosomes were defrosted immediately before use.

[0121] Prior to using the pg160, which was obtained containing 0.01%TWEEN detergent the gp160 was prepared in accord by one of the twofollowing methods:

[0122] 1) Dialysis: Seven ml of pg 160 containing TWEEN was dialyzedacross a SpectraPor membrane with molecular weight cut-off (MWCO) of100,000 daltons against two liters of 0.1M Tris buffered normal saline,pH 8.0 at 4° C. for four days, changing the buffer solution once perday. As an example, in one instance, 10.7 mg of gp160 in 0.1M Trisbuffered saline was used of a stock of 0.54 mg/ml concentration in avolume of 19.8 mls. Next, Empigen BB (stock solution of 30% was added toresult in a final concentration of 1% of Empigen (0.64 ml).

[0123] The proteosomes were added to provide a 1:1 ratio (weight:weight)so that 10.7 mg of 6.7 mg/ml stock in 1.6 ml was added to result in afinal concentration of 0.485 mg/ml of gp160 and proteosomes. Theresulting product was dialyzed across a 1000 MWCO spectraPor 6 or 7membrane for 10 days at 4° C. against Tris buffered saline changing thebuffer daily.

[0124] 2) Centrifugal Dialysis: Centriprep 30 tubes were used tosimultaneously remove the TWEEN and concentrate the gp160 stock from 0.7mg/ml to >4 mg/ml by diluting 15 mls of the 0.7 ml stock with 5 mls ofTris buffered saline to result in a concentration of 0.5 mg/ml. This wascentrifuged at 2,000 g in a Beckman centrifuge for 15 minutes at 4° C.to result in 10 ml of partially concentrated gp160. This was diluted to20 mls and recentrifuged as above to result in 10 ml volume. Theresulting concentrate was rediluted with Tris buffered saline to 30 mlsand recentrifuged as above to result in a final volume of 3.2 mls with agp160 concentration of 4.25 mg/ml (analyzed spectro-photometrically at0.280) and with an estimated 99.999% TWEEN removal and 94% recovery ofgp160. For example, 7.2 mg of gp160 in 0.1M Tris buffered saline wasused of a stock of 4.2 mg/ml concentration in a volume of 1.7 mls. Next,Empigen BB (stock solution of 30% was added to result in a finalconcentration of 1% of Empigen (0.08 ml).

[0125] The proteosomes were added to provide a 1:1 ratio (weight:weight)so that 7.2 mg of 6.7 mg/ml stock in 1.1 ml was added to result in afinal concentration of 2.5 mg/ml of gp160 and proteosomes. The resultingproduct was dialyzed across a 1000 MWCO spectraPor 6 or 7 membrane for10 days at 4° C. against Tris buffered saline changing the buffer daily.

[0126] The pg160 is a much larger than the R32ft disclosed herein. Thegp160 is a transmembrane protein. Furthermore, it naturally formstrimers that make its molecular weight even larger. The antigenicproperties compositions containing gp160 complexed to proteosomes can beenhanced by addition of adjuvants such as alum. It has also beendiscovered that sub micron emulsions enhance immunogenicity. Table 6gives a comparison of ELSA anaylsis of sera from rabits immunized 4times i.m. with 85 μg of gp160 formulated with alum, proteosomes plusalum, or proteosomes plus sub-micron emulsions: TABLE 6 ENHANCED SERUMANTIBODY RESPONSE TO THRE GP160 ANTIGENS INDUCED IN RABBITS BYFORMULATING GP160 WITH PROTEOSOMES PLUS ALUM COMPARED TO GP160 Geometricmean of serum IgG titers Vaccine gp160 gp41 Alex 10* gp160/alum 30,274680 1 gp160/proteosome/ 51,112 565 693 alum gp160/proteosome/ 104,6641,538 200 SME

[0127] Data shows that gp160/proteosome/adjuvant provides vaccine withimproved antigenic effects.

[0128] Leishmania vaccine:

[0129] Mice immunized and then infected with L. major in a murine modelof cutaneous leishmaniasis having a lauryl or lauryl-cysteine conjugatedto the amino terminus was assessed for cell mediated immune response.Vaccines will consist of lauryl or lauryl-cysteine conjugated to aselected synthetic gp63 peptide 467-482 having the structure Gly Asn ValGln Ala Ala Lys Asp Gly Gly Asn Thr Ala Ala Gly Arg The peptidecovalently conjugated to lauryl-cysteine protected against severeleishmania cutaneous lesions with an average of 81% reduction of lesionsin 3 separate experiments. This occurred even when giving thelauryl-cysteinyl-peptide in saline without other adjuvants whereas thecysteinyl-peptide or the peptide without the added lauryl moiety wasineffective. Addition of proteosomes or other peptides did not furtherenhance protection. Proliferative studies were negative. Gene bankanalysis of this peptide revealed a striking homology with a humanintegrin molecule responsible for localization of cellular elements inthe inflammatory process indicating that the parasite may use immunemimicry to avoid host immune defense mechanisms. This peptide maytherefore have wide application in ameliorating pathologic cellularimmune responses caused by other forms of leishmania or other parasitesor bacteria such as mycobacteria where CMI protection is important.

[0130] Proteosomes confer intranasal immunogenicity on formalinizedtoxoid of Staphylococcal Enterotoxin B (SEB) when formulated withproteosomes. In mice anti-SEB respiratory IgA and serum IgG were inducedwhen the complexed compositions in saline were administeredintranasally. The proteosome-toxoid vaccine also showed enhancedimmunogenically when given parenterally. The proteosome-toxoid vaccinewas made by the dialysis method as described. The toxoid and proteosomeswere mixed in the presence of 1% buffered detergent (Empigen) anddialyzed.

[0131] Mice immunized intranasally with proteosome-toxoid vaccines weresignificantly protected (p<0.0117) against systemic challenge with >4LD100 of SEB using the D-galactosamine SEB challenge model. Miceimmunized parenterally with proteosome-toxoid vaccines responded withhigh levels of anti-SEB serum IgG which were further enhanced byadjuvant in with alum. Using the D-galactosamine model, 98% of the 55mice immunized parenterally with these vaccines that induced highanti-SEB serum IgG were protected against parenteral SEB challengewhereas mice immunized with the formalinized toxoid in saline or alumthat had titers <55,000 were significantly less protected.

[0132] As indicated, the methods of the invention are appropriate foruse both with addition of the hydrophobic foot. However, when there is ahydrophobic moiety in or associated with the peptide, it is notnecessary to synthetically add the hydrophobic foot.

[0133] The examples provided herein are for exemplification only, andare not to be construed as suggesting limitation thereto.

1. A construct comprising gp160 and a proteosome.
 2. A composition ofmatter comprising a vaccine which contains as an active agent aconstruct of claim 1 in a pharmaceutically acceptable carrier.
 3. Acomposition of claim 2 containing an adjuvant.
 4. A composition of claim3 wherein the adjuvant is alum.